We propose several approaches to enhance the molecular genetical base for an important experimental system, the purple sea urchin, Strongylocentrotus purpuratus. We plan to generate cultured lines with increased genetic homozygosity in which we can study some remaining gaps in our understanding of sea urchin life cycles, their genetic structure and manipulation and gene transfer technology. Because we can efficiently carry out egg-to-egg culture we have already produced a 4th generation inbred line as a starting point for production of inbred standard lines. We will analyze the level of heterozygosity in these lines by restriction fragment length polymorphism using the large collection of available probes, and techniques established in our labs. A natural though rarely occurring source for animals of increased homozygosity is hermaphroditic individuals several of which are now maintained in culture. We propose to use cold. treatment during gonadogenesis to greatly increase hermaphrodite frequency and hopefully derive a protocol for inducing this condition at will. Another approach we propose is utilization of isogenic twins and quadruplets derived from single fertilized eggs. Since sex determination appears to be subject to an environmental temperature factor in Strongylocentrotus purpuratus, we propose to generate both sexes from sets of isogenic twins, or quadruplets by appropriate temperature regimes during development. Because increased homozygosity produced by the above methods may uncover recessive developmental lethals and other developmental mutations of interest, we will screen embryos from homozygous lines and from X-ray and EMS mutagenized lines for deviations from normal embryonic development. We plan to make available to other investigators animals of homozygous inbred strains with known polymorphism, or any interesting mutational variants that we can verify. As an additional approach to understanding gonadogenesis that could be of practical usefulness we will use genotype chimeras to determine the origin of germ cells in the cleavage stage embryo. The techniques we develop to analyze the genetic structure of homozygous lines will also be employed to follow changes in exogenous DNA integrated in the genome during various stages of the life cycle and to determine when and how germ line transformants obtain the low copy number, stable integrations they display.